Medicament for inhibiting tumour growth

ABSTRACT

The present invention relates to novel betulinic acid derivatives with increased activity for the treatment of carcinomas and HIV diseases, a method for preparing such novel betulinic acid derivatives as well as their use as pharmaceuticals.

BACKGROUND OF THE INVENTION 1. Field of the Invention

The present invention relates to novel betulinic acid derivatives withincreased activity for the treatment of carcinomas and HIV diseases, amethod for preparing such novel betulinic acid derivatives as well astheir use as pharmaceuticals. Like betulinic acid, the compoundsaccording to the invention are suitable for clinical use in men andanimals to inhibit the growth of various tumors (melanomas, sarcomas,lymphomas, squamous carcinomas and other tumors mentioned below) as wellas for the treatment of HIV diseases and—due to their antiphlogisticactivity—unspecific inflammatory diseases.

SUMMARY OF THE INVENTION

The invention relates to novel betulic acid derivatives having thegeneral formula (I)

wherein R₁ represents a hydroxy group, an amino group, a protectedhydroxy group or a protected amino group. Suitable protective groups areknown from the prior art, e.g., from Chapters 2 and 7 of “ProtectiveGroups in Organic Synthesis”, T. W. Greene and P. G. M. Wuts, 3^(rd)Edition, John Wiley & Sons, Inc. (1999), that disclosure, thus, beingincorporated in the present description by reference; and R₂=

In particular, the present invention relates to novel betulic acidderivatives having the general formula (I) as indicated above, whereinR₁ represents a hydroxy group, an amino group or one of the followingprotected hydroxy or amino groups:

R₂ is as defined above.

The compounds according to the invention are novel betulic acidderivatives with increased activity and better solubility in polarsolvents, and hence markedly improved application options. It goeswithout saying that any possible salts and inclusion compounds of thecompound according to the invention are covered by the aforementioneddefinition of general formula (I).

The present invention further relates to a method for preparingcompounds of the general formula (I), wherein a betulic acid halide,particularly a betulic acid chloride, appropriately protected by thesubstituent R₁ is reacted with an alcohol or amine appropriatelysubstituted to provide the substituent R₂. If desired, the thus obtainedcompound of the general formula (I) wherein R₁ represents a protectedhydroxy or amino group may then be deprotected, by measures known fromthe prior art, as a function of the selected protective group to providea compound of the general formula (I) wherein R₁ represents hydroxy oramino.

The natural substance betulic acid, which serves as the starting andcomparative compound for the compounds according to the invention, is atriterpene that was isolated already at the beginning of the lastcentury. The name is derived from the respective alcohol betulin, aningredient of birch bark, where it occurs in large quantities. Betulicacid exhibits anti-malaria, anti-inflammatory, anti-HIV and anti-tumoreffects, which have been described in a great number of differentpublications. Used as a chemotherapeutic, betulic acid induces theso-called programmed cell death, also referred to as apoptosis, in tumorcells of various origins (e.g., melanoma cells). In melanomas,antiproliferative effects were detected in human mouse models both invitro and in vivo. Particularly in melanoma cells, betulic acid appearsto specifically induce an apoptotic cell destruction in tumor cells,while melanocytes are largely resistant to this substance. To date,hardly any data on possible synergistic effects between betulic acid andother cytostatics have been available. The molecular action mechanismsin infantile tumors (Ewing sarcoma, medulloblastoma) as well as inglioblastomas were particularly well investigated by the research grouparound S. Fulda (children's hospital, Ulm).

Other known effects of betulic acid and its known derivatives, whichhave been well documented in the scientific literature, include itsactivity against HIV viruses, whose replication and receptor bindingthey are able to suppress, as well as their anti-inflammatory activity,which is, for instance, described by way of a mouse-ear inflammationmodel. Due to its antiproliferative action against various tumors(melanomas, neuroectodermal tumors, sarcomas), which has been describedboth in animal experiments and in cell cultures, betulic acid is, thus,an extremely interesting substance for both single and possiblecombination therapies with other cytostatically active substances andcell-death-modulating substances, e.g., antisensoligonucleotides againstvarious anti-apoptotic Bcl-2 family members and, in particular, Bcl-2,Bcl-xL as well as Mcl-1.

Publications on the action mechanism of betulic acid primarily inmelanomas as well as with Ewing sarcoma, glioblastoma andmedulloblastoma have demonstrated that its effects are to a substantialextent caused by the induction of apoptosis on a mitochondrial level. Itis still unclear today what primary points of attack it has within thecell; amongst others neither any binding sites (receptors), if present,nor the initial signal pathways have been sufficiently explored.However, the inventors and other authors were able to demonstrate thatbetulic acid induces apoptosis in malign cells, yet human melanocytesand even normal cells appear to be less sensitive than malign cells.That observation is of interest, above all, also becauseanimal-experimental data in mice have not revealed any remarkabletoxicity. Apart from those data acquired in cell cultures there areclues to the effect that betulic acid is more strongly concentrated intumor tissues than in normal tissues. The induction of various Bcl-2family members both in melanoma cells and in normal melanocytes and insarcoma lines were investigated too. It was found that the expression ofthe anti-apoptotic protein Mcl-1 can be induced within a few hours bybetulic acid. The other examined proteins of this gene family, above allBcl-2 and Bcl-x, as well as the expression of the p53 protein remainedunchanged under that treatment. The data from the scientific literaturehave indicated that the effects of betulic acid do not depend on the p53protein. Since it has been recently shown that Bcl-2 and Bcl-xL are ableto inhibit betulic-acid-induced apoptosis, these observations suggest acombination of betulic acid with Bcl-2 and/or Bcl-xL antagonization,e.g., by antisensoligonucleotide (ASO). The same considerations apply topossible combinations with, e.g., Mcl-1 ASOs. A property of betulic acidof potential clinical relevance is the recent observation that itscytotoxicity is enhanced in low-pH culture medium. The pH is in manytumors lower than in normal tissues (Noda Y. et al, 1997). The sameauthors found that betulic acid is more active against resting cellsthan against cells in their growth phases. This property might be ofadditional relevance in clinical use, since many chemotherapeutics aswell as radiotherapy are less effective against resting and/or acidoticcell populations.

At present, melanomas, neuroectodermal tumors as well as sarcomas andHIV have above all been the most intensively investigated. These aretumors which are particularly difficult to treat and of which primarilythe generalized disease forms offer hardly promising treatment options.In patients suffering from metastasized melanomas, therapeutical optionsare largely limited to a few substances. These include5-(3,3-dimethyl-2-triazenyl)-1-H-imidazole-4-carboxamide (Dacarbazine,DTIC). Dacarbazine still constitutes the most effective monotherapy inmelanoma, exhibiting response rates in the order of about 30%.Combination therapies with other synthetic or recombinant substances,e.g., BCNU, Cisplatine, Tamoxifen, Interferon-alpha and Interleukin-2show higher response rates in some clinical studies. These are, however,limited in time and entail elevated toxicities. Some substances derivedfrom natural products such as, e.g., Adriamycin, Bleomycin, Etoposideand others have been investigated in respect to their activities againstmelanomas and their toxicities. However, in the end none of thesesubstances turned out to be convincing in clinical daily routine.

DESCRIPTION OF ILLUSTRATIVE EXAMPLES

In the following, the method according to the invention will beexplained in more detail by way of some examples, whereby the disclosureof the invention is not limited to these examples.

1) Acetyl betulic acid-2-amino-3-hydroxy-2-hydroxymethyl propyl ester IV(Compound B)

The synthesis of acetyl betulic acid-2-amino-3-hydroxy-2-hydroxymethylpropyl ester IV is performed by departing from betulic acid I via theintermediate stages of acetyl betulic acid II and the respective acidchloride III, by reacting the acid chloride III withtrishydroxymethylaminomethane.

a) Acetyl betulic acid (MW 498.74) II

Two grams of betulic acid I (MW 456.70) in 50 ml acetic anhydride areheated at reflux for 2 hours. After cooling, the reaction is poured intoice water under vigorous stirring, filtered, and the obtained solid iswashed with water until the acetic acid smell has disappeared.

The solid is then heated at reflux in 70% ethanol for 4 hours understirring.

After cooling, the reaction solution is filtered, the mother liquor isslightly concentrated, cooled in an ice bath and filtered once again.Yield: 86%; mp.: 290° C.

b) Acetyl betulic acid chloride (MW 517.18) III

Two grams of acetyl betulic acid II are provided in dry benzene andsupplemented with a 10-fold excess of oxalyl chloride (3.4 ml). Thereaction mixture is stirred for 8 hours under cooling, and the solventas well as excess oxalyl chloride are subsequently evaporated on arotary evaporator. In order to remove any oxalyl chloride residues,another 20 ml of benzene are added and again evaporated under vacuum.

c) Acetyl betulic acid-2-amino-3-hydroxy-2-hydroxymethyl propyl ester(MW 601.86) IV, (Compound B)

The acid chloride obtained from 1 g acetyl betulic acid (about 0.002mol) by the method according to b) is reacted without furtherpurification. To this end, it is dissolved in 35 ml dioxan (dry), andtris(hydroxymethyl)-aminomethane is added in double excess (0.004 mol,0.5 g). After the addition of a spatula tip of DMAP and 3 drops ofpyridine, the reaction mixture is stirred for 2 days at roomtemperature.

After this, the solids are filtered off, the solution is concentrated onthe rotary evaporator and taken up in chloroform. This chloroformsolution is washed free of pyridine with 1% hydrochloric acid, water andsaturated saline solution several times. After drying over Na₂SO₄, thesolvent is removed and the product is purified over a silica gel columnor (and) chromatotron. A chloroform-methanol mixture at a ratio of 10:1was used as an eluant. Yield: 20%, mp: 156° C.

2) Acetyl betulicacid-N-(1,1-bis(hydroxymethyl)-2-hydroxyethyl)formamide (MW 601.86),(Compound C)

The synthesis of acetyl betulinic acidN-(1,1-bis(hydroxymethyl)-2-hydroxyethyl)formamide (compound C) iscarried out in a manner analogous to reaction scheme 1, departing frombetulic acid I via the intermediate stages of acetyl betulic acid II andthe respective acid chloride III, by reacting the acid chloride III withtris(hydroxymethyl)aminomethane.

The acid chloride obtained from 1 g acetyl betulic acid (about 0.002mol) according to the method of 1)b) is reacted without furtherpurification. To this end, it is dissolved in 35 ml anhydrous dioxan,and an equimolar amount of tris-(hydroxymethyl)aminomethane (0.002 mol,0.25 g) is added. After the addition of a spatula tip of DMAP and 3drops of pyridine, the reaction mixture is heated to 80° C. for 8 hrs.After this, the reaction solution is concentrated on the rotaryevaporator and the residue is taken up in chloroform. This chloroformsolution is washed free of pyridine with 1% hydrochloric acid, water andsaturated saline solution several times. After drying over Na₂SO₄, thesolvent is removed and the product is purified over a silica gel columnor (and) chromatotron. A chloroform-methanol mixture at a ratio of 10:1was used as an eluant. Yield: 15%, mp: 184° C.

In the following, the use of the compounds according to the inventionfor the inhibition of tumor growths are described in comparison tobetulic acid (comparative compound A).

Growth Inhibition in Various Tumor Lines

Examples 1 and 2 show direct comparisons between comparative compound A(betulic acid) and a novel derivative (compound B) in paired growthassays (cell count decrease after 3 days after a single application ofthe indicated concentrations of the respective compounds on day 1).

EXAMPLE 1

Comparison of compound (A) (betulic acid) with compound (B) (protectedbetulic acid trisester)—Growth inhibition in melanoma cell line 518A2(obtained from Peter Schrier, Leiden, The Netherlands) after 48 hrs. TheX-axis indicates the concentration in μg/ml of compounds A and B,respectively, the Y-axis gives the cell survival rate in percent of theuntreated controls.

The ED₅₀ for comparative compound (A) is about 5 μg/ml, that forcompound (B) about 0.7 μg/ml.

Compound (B), thus, exhibits a clearly higher activity than comparativecompound (A), i.e., up to 7 times higher.

A similarly enhanced activity is also observed with sarcomas, as shownbelow by way of examples on a cell line.

EXAMPLE 2

Comparison of the efficacy of comparative compound (A) (betulic acid)with that of compound (B) (protected betulic acid trisester) in aliposarcoma line (ATCC HTB-92). The X-axis indicates the concentrationin μg/ml of compounds A and B, respectively, the Y-axis gives the cellsurvival rate in percent of the untreated controls.

The ED₅₀ for comparative compound (A) is about 2.5 μg/ml, that forcompound (B) is about 0.9 μg/ml.

EXAMPLE 3

Comparison of the effects of different betulic acid derivativesaccording to the invention and of the comparative compound (betulicacid), respectively, on various melanoma-sarcoma lines. The data eachrefer to cell survival rates in percent (mean values) as againstuntreated cell cultures.

3a) Comparative Compound (A) (betulic acid)—Effect on Liposarcoma LineATCC HTB-92

Conc. μg/ml survival in % 0.3 98.0 0.6 92.8 1 92.3 1.25 79.5 2.5 49.3 520.8 10 15.7

3b) Compound (B) (Protected Betulic Acid Trimester)—Effect onLiposarcoma Line ATCC HTB-92

Conc. μg/ml survival in % 0.3 89.5 0.6 70.7 1 12.0 1.25 2.9 2.5 3.0 50.0 10 0.0

3c) Compound (B) (Protected Betulic Acid Trisester)—Effect on MelanomaLine 518A2

Conc. μg/ml survival in % 0.3 83.0 0.6 56.3 1 11.3 1.25 10.3 2.5 2.8 50.0 10 0.0

3d) Compound (C) (Protected Betulic Acid Trisester)—Effect on MelanomaLine 518A2 and Liposarcoma Line ATCC HTB-92

Conc. μg/ml survival in % melanoma 0.25 96 0.5 93 1 67.5 Conc. μg/mlsurvival in % liposarcoma 0.3 96 0.6 82 1.0 81Beispiel 4: Preparation of a inclusion compound comprising compound (B)(protected betulinic acid trisester) in HBC(2-hydroxpropyl-beta-cyclodextrin, Sigma, Cat. No. H-107) or in HGC(2-hydroxypropyl-gamma-cyclodextrin, Sigma, Cat. No. H-125).

The respective stock solutions for further dilution are indicated below.Fifty or 100 mg/ml of compound (B) were each dissolved in EtOH. HBC wasseparately dissolved in water or in water plus EtOH. The further mode ofprocedure was basically the same as described in the data sheet suppliedby Sigma. No attempts were made to adjust the pH. The solubility ofcompound (B) in aqueous solutions by means of inclusion in HBC orsimilar compounds definitely remains to be further improved, e.g., ingamma-cyclodextrin. Test #1: compound (B) 50 mg/ml in EtOH dissolved asusual Test #2: compound (B) 5.9 mg/ml in 266 mg/ml HBC in H₂O Test #3:compound (B) 20 mg/ml in 266 mg/ml HBC in 50% EtOH/50% H₂O Test #4:compound (B) 25 mg/ml in 200 mg/ml HGC in 50% EtOH/50% H₂OThe stock solutions indicated above were diluted to 1 mg/ml in EtOH andsubsequently directly added to the culture medium.

4a) Compound (B) (Protected Betulinic Acid Trisester) as InclusionCompound in HBC (2-hydroxpropyl-beta-cyclodextrin) or in HGC(2-hydroxypropyl-gamma-cyclodextrin)—Effect on Melanoma Line 518A2

Survival in percent as with all other data after 3 days of cell cultureafter a single application on day 1

Test #1 Conc. μg/ml Survival in % melanoma 1.0 50 2.0 0.5 3.0 0.0 4.00.0

Test #2 Conc. μg/ml Survival in % melanoma 1.0 65 2.0 25 3.0 0.0 4.0 0.0

Test #3 Conc. μg/ml Survival in % melanoma 1.0 77 2.0 24 3.0 0.0 4.0 0.0

Test #4 Conc. μg/ml Survival in % melanoma 1.0 59 2.0 4 3.0 0.0

4b) Tolerance of compound (B) (protected betulinic acid trisester) asinclusion compound in HGC (2-hydroxypropyl-gamma-cyclodextrin) in aMouse Model

Methodology: A HGC/compound (B) complex dissolved in water wasintravenously injected into the tail vein of pathogen-free femaleC.B.-17 scid/scid (SCID) mice (4-6 weeks old, Harlan Winkelmann,Borchen, Germany). The inclusion compound was prepared as described inExample 4. The alcohol portion was removed by freezedrying of themixture and subsequent reconstitution in water. The total volume perinjection for the experiments indicated below was 200 μl.

Test # 1

Initially, two mice were each treated with 100 μg HGC/compound (B)complex three times in a row on every third day. In case of goodtolerance, the next pair of mice was treated with the next higher doseas indicated in the scheme below.

Scheme:

Group A: 3 times 100 μg HGC/compound (B) complex

Group B 3 times 200 μg HGC/compound (B) complex

Group C 3 times 400 μg HGC/compound (B) complex, then continuation withgroup A treated first:

Group A 3 times 800 μg HGC/compound (B) complex

Group B 3 times 1500 μg HGC/compound (B) complex; then discontinuationbecause of local irritation phenomena.

The tests were discontinued at 1500 μg HGC/compound (B) complex becauseof strong local irritation phenomena in one of two mice, yet without anyapparent systemic toxicity. Overall, an amount of 800 μg HGC/compound(B) complex could be administered per injection per mouse withoutapparent toxicity. At higher concentrations (about >1.5 mg HGC/compound(B) complex per mouse), local irritation phenomena were observed. It isconceivable that slower infusion rates may help avoid such side effects.

Test #2

In a test group of 3 mice, 800 μg HGC/compound (B) complex (in 200 μlvolume) was each administered per mouse every third day, six times in arow. Also this showed no apparent clinically toxic effects. Thesubsequent autopsy did not suggest any macroscopic organ changes.

1.-7. (canceled)
 8. A betulinic acid derivative, salt, or inclusioncompound having a formula (I):

wherein R₁ is a hydroxy group, an amino group, a protected hydroxygroup, or a protected amino group; and R₂ is one of:


9. The compound of claim 8, wherein R₁ is one of the following:


10. The compound of claim 8, further defined as comprised in apharmaceutical composition.
 11. A method for preparing a compound ofclaim 8 comprising reacting a betulic acid halide protected by asubstituent R₁, the substituent R₁ further defined as a protectedhydroxy group or amino group, with an alcohol or amine appropriatelysubstituted to provide the substituent R₂.
 12. The method of claim 11,further comprising deprotecting the protected hydroxy or amino group atR₁ to provide a compound of formula (I) wherein R₁ is a hydroxy or aminogroup.
 13. A method of treating a mammal comprising administering to themammal a compound of claim
 8. 14. The method of claim 13, furtherdefined as a method of inhibiting or treating cancer and/or an HIVdisease.
 15. The method of claim 14, further defined as a method ofinhibiting or treating a tumor.
 16. The method of claim 15, wherein thetumor is further defined as a melanoma or neuroectodermal tumor.
 17. Themethod of claim 14, further defined as a method of inhibiting ortreating a sarcoma.
 18. The method of claim 13, further comprisingadministering to the mammal at least one other substance, furtherdefined as a cytostatically active substance and/orcell-death-modulating substance.
 19. The method of claim 18, wherein theother substance is an antisensoligonucleotide against an anti-apoptoticBcl-2 family member or Mcl-1.
 20. The method of claim 19, wherein theother substance is Bcl-2 or Bcl-xL.